Methods for treating pulmonary fibrosis

ABSTRACT

The present invention provides a method of using arylpiperazine derivatives for treating pulmonary fibrosis. The method comprises a step of administering to a pulmonary fibrosis patient in need thereof an effective amount of a compound of Formula 1, which is an arylpiperazine derivative.

This application is a continuation of U.S. application Ser. No.16/913,414, filed Jun. 26, 2020; which is a continuation ofPCT/US2018/067999, filed Dec. 28, 2018; which claims the benefit of U.S.Provisional Application No. 62/611,501, filed Dec. 28, 2017. Thecontents of the above-identified applications are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention relates to methods of utilizing arylpiperazinederivatives for treating pulmonary fibrosis.

BACKGROUND

Pulmonary fibrosis (PF) is a progressive respiratory disordercharacterized by a scarring and thickening of the lining of the lungsthat causes irreversible loss of ability to transport and exchangeoxygen. As lung tissue scars, it becomes more rigid, making it moredifficult for the lungs to inflate and deflate. When this happens, lessoxygen is transferred into the bloodstream, making it harder to breathe.As PF worsens, a person becomes progressively weaker and short ofbreath, and this damage eventually results in death. When an etiologyfor PF cannot be clearly identified, the condition is termed idiopathicpulmonary fibrosis (IPF).

IPF, a chronic disease that destroys the small interstitial spaceswithin the lungs, is the most common type of diffuse parenchymal lungdisease. IPF is increasingly understood to be the result of anirreversible fibroproliferative and aberrant wound-healing cascade. Thecourse of pulmonary fibrosis and the severity of symptoms can varyconsiderably from person to person. The major signs and symptoms of PFinclude shortness of breath (dyspnea), a dry cough, fatigue, unexplainedweight loss, aching muscles and joints, and widening and rounding of thetips of the fingers or toes (clubbing). Co-morbidities are common withindividuals with PF. The most commonly associated co-morbidities areemphysema (often clinically termed CPFE: combined pulmonary fibrosis andemphysema), pulmonary hypertension (PH), venous thromboembolism, lungcancer, gastroesophageal reflux disease (GERD), cardiovascular disease,diabetes, and neuropsychiatric symptoms such as psychosis, depression,anxiety, and cognitive deficit.

The precise prevalence of IPF worldwide is unknown, but the AmericanLung Association estimates that IPF affects about 140,000 Americansyearly, and about 40,000 people die from it each year. IPF typicallyoccurs in people aged>50 years (range, 40-70 years), and more men thanwomen are affected. Median survival is 2 to 3 years after initialdiagnosis. About two-thirds of IPF patients die within 5 years, and therisk of death increases with age.

Although many advances have been made in recent years, especiallypertaining to the molecular genetics and cell biology of pulmonaryfibrisis (PF), the pathogenesis of PF is still not fully understood.Injuries to epithelial or endothelial cells and disruptions in normalwound healing process contribute to the development of pulmonaryfibrosis (Wynn 2011). Alveoli fibrosis, vascular fibrosis, bloodclotting and coagulation, lung inflammation and respiratory resistanceare morphological hallmarks of pulmonary fibrosis (Wynn 2011). Serotonin(5-HT) and key serotonin receptors have been reported to play a key rolein the pathobiology of PF. Eleveated serotonin levels and expression ofserotonin 5-HT_(2A), 5-HT_(2B) and 5-HT₇ receptors are reported in theepithelial and endothelial cells of PF patients. 5-HT_(2A) receptormodulation reported to regulate blood clotting and coagulation,prolifereation and vasorelaxation. 5-HT_(2B) receptor modulation isreported to play central role in the regulation of fibrosis andproliferation whereas 5-HT₇ is receptor reported to regulateinflammatory cytokines and chemokines (Lofdhal 2016, Mann 2013, and Dees2011).

Despite growing knowledge of the pathobiology of pulmonary fibrosis(PF), the prognosis of patients remains poor. PF is irreversible andcurrently, there is no therapy to stop or significantly delay thedisease progress. Typically, treatment strategies for PF aim to improvequality of life (i.e., relieve disease signs/symptoms) or attempt tolimit further inflammation and scarring. Anti-inflammatory drugs,including corticosteroids and cytotoxic agents, are used even thoughthere is no evidence of a benefit for long-term survival. Pirfenidoneand nintedanib are the two FDA approved drugs for the management of IPF.Both pirfenidone and nintedanib are reported to reduce fibrotic tissueto some extent in the lungs of patients with pulmonary fibrosis, but thetreatment is far from optimal. There is a pressing need for moreeffective and tolerable next generation therapies or treatments that cansignificantly delay the progression of pulmonary fibrosis, if notprovide a cure and improve patients' overall quality of life (QOL).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the schedule of various treatments of the animals duringthe bleomycin-induced IPF study

FIG. 2 shows the survival curves from Day 1-10 (A) and from Day 11-21(B), and body weights (C) of Sham, BLM-induced, and treatment groupanimals. BLM: Bleomycin; Sham: Non-induced animals with the vehicle.*P<0.05 BLM+Veh; as compared to Sham. ** P<0.05; as compared to BLM+Veh.

FIG. 3 shows hemodynamic and cardiac parameters (A-C) and systemicarterial pressures (D-F) measured on Day 21. BLM: Bleomycin; Sham:Non-induced animals with the vehicle. *P<0.05 BLM+Veh; as compared toSham. # P<0.05; as compared to BLM+Veh. ## P<0.05; as compared toBLM+Veh.

FIG. 4 shows respiratory resistance (A) and lung hydroxyproline (B)measured on Day 21. BLM: Bleomycin; Sham: Non-induced animals with thevehicle. *P<0.05, BLM+Veh; as compared to Sham. *** P<0.001; as comparedto Sham. # P<0.05; as compared to BLM+Veh. ## P<0.01; as compared toBLM+Veh.

FIG. 5 shows parameters reflective of pulmonary edema at Day 21including lung weight (A), BALF cell count (B), and BALF total protein(C). BALF: Bronchoalveolar lavage; BLM: Bleomycin; Sham: Non-inducedanimals with the vehicle. **P<0.01, BLM+Veh; as compared to Sham. ***P<0.001; as compared to Sham. # P<0.05; as compared to BLM+Veh.

FIG. 6 shows morphology changes displayed by H&E staining and AshcroftScore (A, C). The collagen deposition is reflected by Masson's trichromestaining (B, D) induced by BLM in rats on Day 21. BLM: Bleomycin; Sham:Non-induced animals with the vehicle. BLM+RP5063 (all animals). ***P<0.001; as compared to Sham. ### P<0.001; as compared to BLM+Veh.

FIG. 7 shows BLM-induced effects on blood oxygen saturation (A) andblood lactate levels (B) measured at Day 21. BLM: Bleomycin; Sham:Non-induced animals with the vehicle. ** P<0.01; as compared to Sham. #P<0.05; as compared to BLM+Veh. ## P<0.01; as compared to BLM+Veh.

FIG. 8 shows BALF cytokines levels on Day 21: MIP (A); MCP1 (B); IL6(C); IP10 (D); and RANTES (E). BLM: Bleomycin; Sham: Non-induced animalswith the vehicle.* P<0.05; as compared to Sham. *** P<0.001; as comparedto Sham. # P<0.05; as compared to BLM+Veh. ## P<0.01; as compared toBLM+Veh. ### P<0.001; as compared to BLM+Veh.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” or “alkanyl” refers to a saturated, branched or straight-chainor cyclic monovalent hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane. Typicalalkyl groups include, but are not limited to methyl; ethyl; propyls suchas propan-1-yl, propan-2yl, cyclopropan-1-yl; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutan-1-yland the like. Preferably, an alkyl group comprises from1-20 carbon atoms, more preferably, from 1 to 10, or 1 to 6, or 1-4carbon atoms.

“Alkenyl” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon double bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkene. The group may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl,cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methy-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien 1-yl, etc.; and the like.

“Alkynyl” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon triple bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such asbut-1-yn-1-yl, but-1-yn3-yl, but-3-yn-1-yl, etc.; and the like.

“Acyl” refers to a radical —C(O)R, where R is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl, as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Acylamino” refers to “amide” as defined herein.

“Alkylamino” means a radical —NHR where R represents an alkyl, orcycloalkyl group as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to, methylamino, ethylamino,1-methylethylamino, cyclohexylamino and the like.

“Alkoxy” refers to a radical —OR where R represents an alkyl, orcycloalkyl group as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to methoxy, ethoxy, propoxy, butoxy,cyclohexyloxy and the like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Alkylsulfonyl” refers to a radical —S(O)₂R where R is an alkyl, orcycloalkyl group as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to, methylsulfonyl, ethylsulfonyl,propylsulfonyl, butylsulfonyl, and the like.

“Alkylsulfinyl” refers to a radical —S(O)R where R is an alkyl, orcycloalkyl group as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to, methylsulfinyl, ethylsulfinyl,propylsulfinyl, butylsulfinyl, and the like.

“Alkylthio” refers to a radical —SR where R is an alkyl or cycloalkylgroup as defined/herein that may be optionally substituted by one ormore substituents as defined herein. Representative examples include,but are not limited to methylthio, ethylthio, propylthio, butylthio, andthe like.

“Amide” or “acylamino” refers to a radical —NR′C(O)R″, where R′ and R″are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as definedherein that may be optionally substituted by one or more substituents asdefined herein. Representative examples include, but are not limited to,formylamino acetylamino, cyclohexylcarbonylamino,cyclohexylmethylcarbonyl-amino, benzoylamino, benzylcarbonylamino andthe like.

“Amino” refers to the radical —NH₂.

“Aryl” refers to a monovalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorine, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleidene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. Preferable, anaryl group comprises from 6 to 20 carbon atoms, more preferably, between6 to 12 carbon atoms.

“Arylalkyl” refers to an acyclic alkyl in which one of the hydrogenatoms bonded to a carbon atom, typically a terminal or sp³ carbon atom,is replaced with an aryl group. Typically arylalkyl groups include, butnot limited to, benzyl, 2-phenylethan-1-yl, naphthylmethyl,2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Preferably, an arylalkyl group is (C₆-C₃₀)arylalkyl, e.g., thealkyl moiety of the arylalkyl group is (C₁-C₁₀) and the aryl moiety is(C₆-C₂₀), more preferably, an arylalkyl group is (C₆-C₂₀) arylalkyl,e.g., the alkyl moiety of the arylalkyl group is (C₁-C₈) and the arylmoiety is (C₆-C₁₂).

“Arylalkoxy” refers to an —O-arylalkyl radical where arylalkyl is asdefined herein that may be optionally substituted by one or moresubstituents as defined herein.

“Aryloxycarbonyl” refers to radical —C(O)—O-aryl where aryl is definedherein that may be optionally substituted by one or more substituents asdefined herein.

“Carbamoyl” refers to the radical —C(O)NRR where each R group isindependently, hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined hereinthat may be optionally substituted by one or more substituents asdefined herein.

“Carbamate” refers to a radical —NR′C(O)OR″, where R′ and R″ are eachindependently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined hereinthat may be optionally substituted by one or more substituents asdefined herein. Representative examples include, but are not limited to,methylcarbamate (—NHC(O)OCH₃), ethylcarbamate (—NHC(O)OCH₂CH₃),benzylcarbamate (—NHC(O)OCH₂C₆H₅), and the like.

“Carbonate” refers to a radical —OC(O)OR, where R is alkyl, cycloalkyl,cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl, as defined herein that may be optionally substituted byone or more substituents as defined herein. Representative examplesinclude, but are not limited to, methyl carbonate (—C(O)OCH₃),cyclohexyl carbonate (—C(O)OC₆Hu), phenyl carbonate (—C(O)OC₆H₅), benzylcarbonate (—C(O)OCH₂C₆H), and the like.

“Carboxy” means the radical —C(O)OH.

“Cyano” means the radical —CN.

“Cycloalkyl” refers to a substituted or unsubstituted cylic alkylradical. Typical cycloalkyl groups include, but are not limited to,groups derived from cyclopropane, cyclobutane, cyclopentane,cyclohexane, and the like. In a preferred embodiment, the cycloalkylgroup is (C₃-C₁₀) cycloalkyl, more preferably (C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” refers to a saturated or unsaturated cyclic alkylradical in which one or more carbon atoms (and any associated hydrogenatoms) are independently replaced with the same or different heteroatom.Typical heteroatoms to replace the carbon atom(s) include, but are notlimited to, N, P, O, S, Si, etc. Where a specific level of saturation isintended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl”is used. Typical cycloheteroalkyl groups include, but are not limitedto, groups derived from epoxides, imidazolidine, morpholine, piperazine,piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like.

“Halogen” means fluoro, chloro, bromo, or iodo.

“Heteroaryl” refers to a monovalent heteroaromatic radical derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, trazole, xanthene, and the like. Preferably, the heteroarylgroup is between 5-20 membered heteroaryl, with 5-10 membered heteroarylbeing particularly preferred. Preferred heteroaryl groups are thosederived from thiophene, pyrrole, benzothiophene, benzofuran, indole,pyridine, quinoline, imidazole, oxazole and pyrazine.

“Heteroaryloxycarbonyl” refers to a radical —C(O)—OR where R isheteroaryl as defined that may be optionally substituted by one or moresubstituents as defined herein.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp3carbon atom, is replaced with a heteroaryl group. Preferably, theheteroarylalkyl radical is a 6-30 carbon membered heteroarylalkyl, e.g.,the alkyl moiety of the heteroarylalkyl is 1-10 membered and theheteroaryl moiety is a 5-20 membered heteroaryl, more preferably, a 6-20membered heteroarylalkyl, e.g., the alkyl moiety of the heteroarylalkylis 1-8 membered and the heteroaryl moiety is a 5-12 membered heteroaryl.

“Hydroxy” means the radical —OH.

“Oxo” means the divalent radical ═O.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention, which is pharmaceutically acceptable and possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentane propionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Phosphate” refers to a radical —OP(O)(OR′)(OR″), where R′ and R″ areeach independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined hereinthat may be optionally substituted by one or more substituents asdefined herein.

“Phosphonate” refers to a radical —P(O)(OR′)(OR″), where R′ and R″ areeach independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined hereinthat may be optionally substituted by one or more substituents asdefined herein.

“Preventing” or “Prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

“Racemate” refers to an equimolar mixture of enantiomers of a chiralmolecule.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituents(s).Typical substituents include, but are not limited to, —X, —R⁵⁴, —O—, ═O,—OR⁵⁴, —SR⁵⁴, —S, ═S, —NR⁵⁴R⁵⁵, ═NR⁵⁴, —CX₃, —CF₃, —CN, —OCN, —SCN, —NO,—NO₂, ═N, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂OR⁵⁴, —OS(O)₂O³¹, —OS(O)₂R⁵⁴,—P(O)(O—)₂, —P(O)(OR)⁵⁴)(O³¹), —OP(O)(OR⁵⁴)(OR⁵⁵), —C(O)R⁵⁴, —C(S)R⁵⁴,—C(O)OR⁵⁴, —C(O)NR⁵⁴R⁵⁵, —C(O)O⁻, —C(S)OR⁵⁴, —NR⁵⁶C(O)NR⁵⁴R⁵⁵,—NRC(S)NR⁵⁴R⁵⁵, —NR⁵⁷C(NR⁵⁶)NR⁵⁴R⁵⁵, and —C(NR⁵⁶)NR⁵⁴R⁵⁵, where each Xis independently a halogen; each R⁵⁴, R⁵⁵, R⁵⁶ and R⁵⁷ are independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, —NR⁵⁸R⁵⁹, —C(O)R⁵⁸ or —S(O)₂R⁵⁸ oroptionally R⁵⁸ and R⁵⁹ together with the atom to which they are bothattached form a cycloheteroalkyl or substituted cycloheteroalkyl ring;and R⁵⁸ and R⁵⁹ are independently hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl.

“Sulfate” refers to a radical —OS(O)(O)OR, where R is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl, as defined herein that may be optionally substituted byone or more substituents as defined herein.

“Sulfonamide” refers to a radical —S(OXO)NR′R″, where R′ and R″ areindependently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined hereinthat may be optionally substituted by one or more substituents asdefined herein or optionally R′ and R″ together with the atom to whichthey are both attached form a cycloheteroalkyl or substitutedcycloheteroalkyl ring. Representative examples include but not limitedto azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl,4-(NR′″)-piperazinyl or imidazolyl group wherein said group may beoptionally substituted by one or more substituents as defined herein.R′″ hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein that may beoptionally substituted by one or more substituents as defined herein.

“Sulfonate” refers to a radical —S(O)(O)OR, where R is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl, as defined herein that may be optionally substituted byone or more substituents as defined herein.

“Thio” means the radical —SH.

“Thioether” refers to a radical —SR, where R is alkyl, cycloalkyl,cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl, as defined herein that may be optionally substituted byone or more substituents as defined herein.

“Treating” or “Treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the patient. In yet another embodiment, “treating” or“treatment” refers to inhibiting the disease or disorder, eitherphysically (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating a disease, is sufficient toaffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the compound, the disease and is severityand the age, weight, etc., of the patient to be treated, and can bedetermined by one of skill in the art without undue experimentation.

The present invention is directed to a method for treating pulmonaryfibrosis.

Compounds Useful for the Invention

Compounds of Formula (I) are useful for the present invention:

wherein:

-   A is —O—(CH₂)_(n)—, —(CH₂)—, —S—(CH₂)_(n)—, —S(OXO)—(CH₂)_(n)—,    —NH—(CH₂)_(n)—, —CH₂—O—(CH₂)_(n)—, —(CH₂)_(n)—O—CH₂—CH₂—,    —CH₂—S—(CH₂)_(n)—, —(CH₂)_(n)—S—CH₂—CH₂—, —CH₂—S(OXO)—(CH₂)_(n)—,    —(CH₂)_(n)—S(OXO)—CH₂—CH₂—, —O—C(O)—(CH₂)_(n)—, —S—C(O)—(CH₂)_(n)—,    —NH—C(O)—(CH₂)_(n)—, —CH₂—C(O)—O—(CH₂)_(n)—,    —CH₂—C(O)—NH—(CH₂)_(n)—, —CH₂—C(O)—S—(CH₂)_(n)—,    —(CH₂)_(n)—C(O)—O—CH₂—CH₂—, —(CH₂)_(n)—C(O)—NH—CH₂—CH₂—,    —(CH₂)_(n)—C(O)—S—CH₂—CH₂—, —CH₂—O—C(O)—(CH₂)_(n)—,    —CH₂—NH—C(O)—(CH₂)_(n)—, —CH₂—S—C(O)—(CH₂)_(n)—,    —(CH₂)_(n)—O—C(O)—CH₂—CH₂—, (CH₂)_(n)—NH—C(O)—CH₂—CH₂—, or    (CH₂)_(n)—S—C(O)—CH₂—CH₂—, wherein n is an integer from 1 to 7,    preferably n is 2 to 5, for example n is 4;-   B is O, S, S(OXO), or NR⁵; and-   each of R, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently hydrogen,    alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,    substituted arylalkyl, cycloalkyl, substituted cycloalkyl,    cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl,    substituted heteroaryl, heteroarylalkyl, substituted    heteroarylalkyl, acylalkyloxycarbonyl, acyloxyalkyloxycarbonyl,    acylalkyloxycarbonylamino, acyloxyalkyloxycarbonylamino, alkoxy,    alkoxycarbonyl, alkoxycarbonylalkoxy, alkoxycarbonyllalkylamino,    alkylsulfinyl, alkylsulfonyl, alkylthio, amino, alkylamino,    arylalkylamino, dialkylamino, arylalkoxy, arylalkoxycarbonylalkoxy,    arylalkoxycarbonylalkylamino, aryloxycarbonyl,    aryloxycarbonylalkoxy, aryloxycarbonylalkylamino, carboxy,    carbamoyl, carbamate, carbonate, cyano, halo, heteroaryloxycarbonyl,    hydroxy, phosphate, phosphonate, sulfate, sulfonate, or sulfonamide,    wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ and A may optionally be    substituted with isotopes that include, but not limited to ²H    (deuterium), ³H (tritium), ¹³C, ³⁶Cl, ¹⁸F, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P,    and ³⁵S; with ²H (deuterium) being preferred;-   or a pharmaceutically acceptable salt, racemate or diastereomeric    mixtures thereof.

In one aspect of the invention, A is —O—(CH₂)_(n)—.

In one aspect of the invention, A is —(CH₂)_(n)—.

In another aspect of the invention, A is —S—(CH₂)_(n)—,—CH₂—O—(CH₂)_(n)—, —(CH₂)_(n)—O—CH₂—CH₂—, —CH₂—S—(CH₂)_(n)—, or—(CH₂)_(n)—S—CH₂—CH₂—; with A being —O—(CH₂)_(n)— preferred.

In another aspect of the invention, A is —NH—C(O)—(CH₂)_(n)—,—CH₂—NH—C(O)—(CH₂)_(n)—, —CH₂—C(O)—NH—(CH₂)_(n)— or—(CH₂)_(n)—C(O)—NH—CH₂—CH₂—.

In another aspect of the invention, B is O.

In another aspect of the invention, R³, R⁴, R⁶, R⁶, and R⁸ are H.

In a preferred embodiment, A is —O—(CH₂)_(n)— or —NH—C(O)—(CH₂)_(n)—,n=2-5.

In a preferred embodiment, B is O.

In a preferred embodiment, R³, R⁴, R⁶, R⁶, and R⁸ are H.

In a preferred embodiment, each of R¹ and R² is independently H, halogen(e.g., chloro), haloalkyl, or alkoxy (e.g., methoxy or ethoxy);preferably halogen or alkoxy.

Preferred compounds of Formula I include, for example, the followingCompounds A-D and their hydrochloride salt.

The compounds useful for the present invention further pertain toenantiomerically isolated compounds of Formula I. The isolatedenantiomeric forms of the compounds of Formula I are substantially freefrom one another (i.e., in enantiomeric excess). In other words, the “R”forms of the compounds are substantially free from the “S” forms of thecompounds and are, thus, in enantiomeric excess of the “R” forms.Conversely, “S” forms of the compounds are substantially free of “R”forms of the compounds and are, thus, in enantiomeric excess of the “S”forms. In one embodiment of the invention, the isolated enantiomericcompounds are at least about in 80% enantiomeric excess. Thus, forexample, the compounds are at least about 90% enantiomeric excess,preferably at least about 95% enantiomeric excess, more preferably atleast about 97% enantiomeric excess., or even more preferably, at least99% or greater than 99% enantiomeric excess.

Formula I compounds can be synthesized according U.S. Pat. No.8,188,076, which is incorporated herewith in its entirety.

Method of Treating Pulmonary Fibrosis

The present invention is directed to a method for treating pulmonaryfibrosis (PF) and idiopathic pulmonary fibrosis (IPF). When the causefor PF cannot be clearly identified, the condition is termed IPF.Although the cause of PF and IPF may be different, the signs andsymptoms of PF and IPF are the same, and the present invention iseffective to treat PF and IPF, regardless the cause of the disease. Themethod comprises the step of administering an effective amount of acompound of Formula I to a patient who is suffering from pulmonaryfibrosis. Formula I compounds can lower fibrosis in the pulmonary artery(the blood vessel that leads from the heart to the lungs) or alveoli ofa patient and treat pulmonary fibrosis. The treatment can also reducedisease complications, such as lung inflammation, shortness of breath,pain crisis, pneumonia, and increase survival.

In one embodiment, the method treats pulmonary fibrosis in a subjectwith chronic obstructive pulmonary disease (COPD), with pulmonaryarterial hypertension (PAH), with sickle cell disease (SCD), withscleroderma or with lung cancer.

In one embodiment, the method treats comorbid mental illnesses such aspsychosis, depression, and mood symptoms in patients with pulmonaryfibrosis. In another embodiment, the method treats anxiety in patientswith pulmonaryfibrosis.

When used to treat pulmonary fibrosis, one or more compound of Formula Ican be administered alone, or in combination with other agents, to apatient. The patient may be an animal, preferably a mammal, and morepreferably a human.

Formula I compounds are preferably administered orally. Formula Icompounds may also be administered by any other convenient route, forexample, by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.). Administration can be systemic or local.Various delivery systems are known, (e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc.) that can be used toadminister a compound and/or composition of the invention. Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intranasal, intracerebral, intravabinal,transdermal, rectally, by inhalation, or topically, particularly to theears, nose, eyes or skin. Inhalation or transdermal administration maybe preferred for young children.

Formula I compounds can be delivered via sustained release systems,preferably oral sustained release systems. In one embodiment, a pump maybe used (see, Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng.14:201; Saudek et al., 1989, N. Engl. J. Med. 321:574).

In one embodiment, polymeric materials can be used (see “MedicalApplications of Controlled Release,” Langer and Wise (eds.), Wiley, NewYork (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. MacromolChem. 23:61; see also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71:105). Ina preferred embodiment, polymeric materials are used for oral sustainedrelease delivery. Preferred polymers include sodiumcarboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred,hydroxypropylmethylcellulose). Other preferred cellulose ethers havebeen described in the art (Bamba et al., Int. J. Pharm., 1979, 2, 307).

In one embodiment, enteric-coated preparations can be used for oralsustained release administration. Preferred coating materials includepolymers with a pH-dependent solubility (i.e., pH-controlled release),polymers with a slow or pH-dependent rate of swelling, dissolution orerosion (i.e., time controlled release), polymers that are degraded byenzymes (i.e., enzyme controlled release) and polymers that form firmlayers that are destroyed by an increase in pressure (i.e.,pressure-controlled release).

In still another embodiment, osmotic delivery systems are used for oralsustained release administration (Verma et al., Drug Dev. Ind. Pharm.,2000, 26:695-708). In a preferred embodiment, OROS® osmotic deliverysystems are used for oral sustained release delivery devices (See forexample, Theeuwes et al., U.S. Pat. No. 3,845,770; and Theeuwes et al,U.S. Pat. No. 3,916,899).

In yet another embodiment, a controlled-release system can be placed inproximity of the target of the compounds and/or composition of theinvention, thus requiring only a fraction of the systemic dose (See,e.g., Goodson, in “Medical Applications of Controlled Release,” supra,vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussedin Langer, 1990, Science 249:1527-1533 may also be used.

Formula I compounds may be cleaved either chemically and/orenzymatically. One or more enzymes present in the stomach, intestinallumen, intestinal tissue, blood, liver, brain or any other suitabletissue of a mammal may enzymatically cleave the compounds and/orcompositions of the invention.

Pharmaceutical Formulation of the Invention

The present invention is directed to a pharmaceutical formulation fortreating pulmonary fibrosis. The pharmaceutical formulation contains atherapeutically effective amount of one or more compounds of Formula I,preferably in purified form, together with a suitable amount of apharmaceutically acceptable vehicle. When administered to a patient, thepharmaceutical formulation is preferably sterile. Water is a preferredvehicle when the compound of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid vehicles, particularly forinjectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present agents, or pH buffering agents.In addition, auxiliary, stabilizing, thickening, lubricating andcoloring agents may be used.

Pharmaceutical compositions comprising a compound of the invention maybe manufactured by means of conventional mixing, dissolving,granulating, levigating, and emulsifying, encapsulating, entrapping orlyophilizing process. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries, which facilitateprocessing of compounds of the invention into preparations which can beused pharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, and capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable vehicle is acapsule (see e.g., Grosswald et al., U.S. Pat. No. 5,698,155). Otherexamples of suitable pharmaceutical vehicles have been described in theart (see Remington's Pharmaceutical Sciences, Philadelphia College ofPharmacy and Science, 17^(th) Edition, 1985). Preferred compositions ofthe invention are formulated for oral delivery, particularly for oralsustained release administration.

Compositions for oral delivery may be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups or elixirs, for example. Orally administered compositions maycontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry coloring agents and preservingagents to provide a pharmaceutically palatable preparation. Moreover,where in tablet or pill form, the compositions may be coated to delaydisintegration and absorption in the gastrointestinal tract, therebyproviding a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds ofthe invention. In these later platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving compound, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time delay material such as glycerol monostearate orglycerol stearate may also be used. Oral compositions can includestandard vehicles such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Such vehiclesare preferably of pharmaceutical grade.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols(e.g., polyethylene glycol) oils, alcohols, slightly acidic buffersbetween pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at betweenabout mM to about 50 mM) etc. Additionally, flavoring agents,preservatives, coloring agents, bile salts, acylcarnitines and the likemay be added.

Compositions for administration via other routes may also becontemplated. For buccal administration, the compositions may take theform of tablets, lozenges, etc. formulated in conventional manner.Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include a compoundof the invention with a pharmaceutically acceptable vehicle. Preferably,the pharmaceutically acceptable vehicle is a liquid such as alcohol,water, polyethylene glycol or a perfluorocarbon. Optionally, anothermaterial may be added to alter the aerosol properties of the solution orsuspension of compounds of the invention. Preferably, this material isliquid such as alcohol, glycol, polyglycol or fatty acid. Other methodsof formulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (see, e.g.,Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).A compound of the invention may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa, butter or otherglycerides. In addition to the formulations described previously, acompound of the invention may also be formulated as depot preparation.Such long acting formulations may be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, a compound of the invention may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

Dosage for the Treatment

The amount of Formula I compound administered is dependent on, amongother factors, the subject being treated, and the weight of the subject,the severity of the affliction, the manner of administration and thejudgment of the prescribing physician. For example, the dosage may bedelivered in a pharmaceutical composition by a single administration, bymultiple applications or controlled release. In one embodiment, thecompounds of the invention are delivered by oral sustained releaseadministration. In one embodiment, the compounds of the invention areadministered twice per day, and preferably, once per day. Dosing may berepeated intermittently, may be provided alone or in combination withother drugs, and may continue as long as required for effectivetreatment of the disease state or disorder.

The compounds of Formula I may be administered in the range 0.1 mg to500 mg, preferably 1 mg to 100 mg per day, such as 5 mg, 10 mg, 15 mg,20 mg, 25 mg, 35 mg or 50 mg per day, and preferably 10 mg per day.

Combination Therapy

In certain embodiments of the present invention, the compounds of theinvention can be used in combination therapy with at least one othertherapeutic agent. Formula I compounds and the therapeutic agent can actadditively or synergistically. In one embodiment, Formula I compound isadministered concurrently with the administration of another therapeuticagent, which can be part of the same composition of Formula I compound.In another embodiment, a composition comprising a compound of theinvention is administered prior or subsequent to administration ofanother therapeutic agent.

The present invention is effective for treating pulmonary fibrosis.Compounds of Formula I have potent binding affinity at the serotonin5-HT_(2A) receptor (compound A, Ki=2.5 nM, see Example 1), 5-HT_(2B)receptor (compound A, Ki=0.19 nM, see Example 1), and 5-HT₇ receptor(compound A, Ki=2.7 nM, see Example 1). In addition, Compounds ofFormula I exhibit partial agonist activities for the key subtypes ofdopamine (D2) and serotonin (5-HT_(1A)), and antagonist activity at theserotonin 5-HT₆ receptors. Furthermore, compounds of Formula I (compoundA) demonstrated efficacy for treating pulmonary fibrosis in bleomycininduced pulmonary fibrosis rat model (Example 2).

The invention is illustrated by the following examples.

EXAMPLES Example 1. In Vitro Pharmacology Results

Two arylpiperazine derivatives of Formula (I), Compound A and CompoundB, were tested in the in vitro pharmacological assays to evaluate theiractivities for dopamine, D_(2S), serotonin, 5-HT_(1A), 5-HT_(2A),5-HT_(2B), 5-HT₆, and 5-HT₇ receptors. The in vitro assay protocols andliterature references are described herein.

Dopamine, D_(2S) Radioligand Binding Assay Materials and Methods:

Receptor Source: Human recombinant D_(2S) expressed mammalian cellsRadioligand: [³H]Spiperone (20-60 Ci/mmol) or [3H]-7-hydroxy DPAT, 1.0nM

Control Compound: Haloperidol or Chlorpromazine

Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mM EDTA for 60minutes at 25 C. The reaction was terminated by rapid vacuum filtrationonto glass fiber filters. Radioactivity trapped onto the filters wasdetermined and compared to control values in order to ascertain anyinteractions of test compounds with the cloned dopamine—D₂ short bindingsite (Literature Reference: Jarvis, K. R. et al. Journal of ReceptorResearch 1993, 13(1-4), 573-590; Gundlach, A. L. et al. Life Sciences1984, 35, 1981-1988.)

Serotonin, 5HT_(1A) Radioligand Binding Assay Materials and Methods:

Receptor Source: Human recombinant 5-HT_(1A) expressed mammalian cells

Radioligand: [³H]-8-OH-DPAT (221 Ci/mmol) Control Compound: 8-OH-DPAT

Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.4) containing mM MgSO₄, 0.5 mM EDTA and 0.1% Ascorbic acid at roomtemperature for 1 hour. The reaction was terminated by rapid vacuumfiltration onto glass fiber filters. Radioactivity trapped onto thefilters was determined and compared to control values in order toascertain any interactions of test compounds with the clonedserotonin-5HT_(1A) binding site (Literature Reference: Hoyer, D. et al.Eur. Journal Pharmacol. 1985, 118, 13-23; Schoeffler, P. and Hoyer, D.Naunyn-Schmiedeberg's Arch. Pharmac. 1989, 340, 135-138)

Serotonin, 5HT_(2A) Radioligand Binding Assay Materials and Methods:

Receptor Source: Human Cortex or Human recombinant 5-HT_(2A) expressedmammalian cells

Radioligand: [³H]-Ketanserin (60-90 Ci/mmol) Control Compound:Ketanserin

Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.6) at room temperature for 90 minutes. The reaction was terminatedby rapid vacuum filtration onto glass fiber filters. Radioactivitytrapped onto the filters was determined and compared to control valuesin order to ascertain any interactions of test compounds with theserotonin-5HT_(2A) binding site (Literature Reference: Leysen, J. E. etal. Mol. Pharmacol. 1982, 21, 301-314; Martin, G. R. and Humphrey, P. P.A. Neuropharmacol. 1994, 33(3/4), 261-273.)

Serotonin, 5HT_(2S) Radioligand Binding Assay Materials and Methods:

Receptor Source: Human recombinant 5-HT_(2B) expressed CHO-K1 cellsRadioligand: 1.20 nM [3H] Lysergic acid diethylamide (LSD)

Control Compound: Ketanserin

Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.6) at room temperature for 90 minutes. The reaction was terminatedby rapid vacuum filtration onto glass fiber filters. Radioactivitytrapped onto the filters was determined and compared to control valuesin order to ascertain any interactions of test compounds with theserotonin-5HT_(2B) binding site.

Serotonin, 5HT₆ Radioligand Binding Assay Materials and Methods:

Receptor Source: Human recombinant 5-HT₆ expressed mammalian cells

Radioligand: [125I] SB258585, 15 nM or [³H]LSD, 2 nM

Control Compound: Methiothepin or serotoninIncubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.4) containing 10 mM MgSO₄, 0.5 mM EDTA and 0.1% Ascorbic acid atroom temperature for 1 hour. The reaction was terminated by rapid vacuumfiltration onto glass fiber filters. Radioactivity trapped onto thefilters was determined and compared to control values in order toascertain any interactions of test compounds with the clonedserotonin-5HT₆ binding site (Literature Reference: Gonzalo, R., et al.,Br. J. Pharmacol., 2006 (148), 1133-1143).

Serotonin, 5HT₇ Radioligand Binding Assay Materials and Methods:

Receptor Source: Human recombinant 5-HT₇ expressed CHO cellsRadioligand: [3H] Lysergic acid diethylamide (LSD), 4 nM

Control Compound: Serotonin

Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl(pH 7.6) at room temperature for 90 minutes. The reaction was terminatedby rapid vacuum filtration onto glass fiber filters. Radioactivitytrapped onto the filters was determined and compared to control valuesin order to ascertain any interactions of test compounds with theserotonin-5HT₇ binding site

The radioligand binding assays were carried out at six to 10 differentconcentrations and the test concentrations were 0.1 nM, 0.3 nM, 1 nM, 10nm, 30 nM, 100 nM, 300 nM and 1000 nM.

The in vitro pharmacological activities of the selected compounds A andB using radioligand binding assays are reported in the following table.Compound A(brilaroxazine)=6-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(4H)-onehydrochloride. CompoundB=6-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(-4H)-onehydrochloride.

Compound Assay Ki A D2S 0.62 nM A 5-HT1A 1.50 nM A 5-HT2A 2.50 nM A5-HT2B 0.19 nM A 5-HT6   51 nM A 5-HT7 2.70 nM B D2S 0.30 nM B 5-HT1A0.65 nM B 5-HT2A  118 nM

Example 2. Evaluation of the Effects of Compound A (RP5063) in theBleomycin (BLM) Induced Pulmonary Fibrosis Rat Model Materials andMethods

Study Animals: The investigation involved 34 male Sprague Dawley rats(weights: 275-300 g; ages: 8 weeks; Charles River Laboratories, Quebec,Canada) randomized for equal distribution according to their body weightinto four groups. Study Design: The objective of this parallel-designstudy was to evaluate the effectiveness of Compound A (RP5063) startedon Day 1 and on Day 10 following BLM-induction on the functional,histologic, and pathophysiologic parameters in the BLM-induced model.

Study Methods: On Day 0, animals in Group 1 (Sham; N=5) received oneintratracheal administration of the vehicle, 0.250 mL of vehicle 0.9%saline solution (FIG. 1). Animals in Groups 2 to 4 (n=9, 10, 10,respectively) received a single intratracheal instillation of 0.250 mL(5 U/kg-3.33 mg/kg) of bleomycin sulfate (Cayman Chemicals, Ann Arbor,Mich.) solution. From Day 1 to 10, the vehicle was administered toGroups 1, 2, and 4. Compound A (RP5063) 15 mg/kg per gavage twice-daily(b.i.d.) was administered to Group 3 as treatment from Day 1 to 20(Group 3, RPT). The vehicle administration was continued to Group 1(Sham) and Group 2 (BLM) until Day 20. From Day 10 to 20, the Group 4received 15 mg/kg per gavage twice-daily (b.i.d.) Compound A (RP5063) asan intervention treatment (Group 4, RPI). During the treatment period,food and water were provided ad libitum to the rats. On each day,animals were monitored for behavior, general health status, andsurvival. Body weight and food intake were also measured.

The solution used was prepared by dissolving 750 mg of Compound A(RP5063) in 500 mL of sterile 5% glucose solution to obtain a solutionof 1.5 mg/mL. For bleomycin, 50 mg of drug was weighed and dissolved in12.5 mL of sterile 0.9% Saline solution was added to obtain a solutionof 4 mg/mL. The vehicle was created by dissolving 50 g of glucose in 1 Lof water to result in a 5% solution.

On Day 21, animals were anesthetized and instrumented. Hemodynamicparameters (systemic arterial blood pressure, heart rate, and oxygensaturation) were recorded continuously for at least 5 minutes. At theend of the recording, a blood sample was collected. After the animal wasexsanguinated, the pulmonary circulation was flushed with 0.9% NaCl, andtissues (lungs, trachea, and heart) were harvested altogether from thethoracic cavity for further analysis.

Parameters Measured on Surgery Day: Cardiac activity was recordedcontinuously during the surgical procedure. Cardiac activity wasmonitored using three electrocardiographic (ECG) contact electrodesplaced in a lead-I/II configuration and connected to an IsoDam8differential amplifier. Heart rate (HR) was recorded using duplicatesystems: from the ECG records (RR-intervals) and using an N-595 pulseoximeter attached to the left front paw of the animal. The heart ratevalues derived from the pulse oximeter were measured in beat per minutes(bpm) using cursor readings in Clampfit 10.2.0.14CA. Blood oxygensaturation (SpO) was measured using a pulse oximeter signal attached tothe left front paw of the animal.

Systemic arterial blood pressure (SAP) was monitored continuously usingan intra-arterial fluid-filled catheter connected to a pressuretransducer, with diastolic and systolic pressures values measured in mmHg. Calculation of mean SAP (mSAP) and pulse pressure (PP) used thefollowing formulas: 1−mSAP=diastolic systemic pressure+([systolicsystemic pressure−diastolic systemic pressure]/3); and 2−PP=systolicsystemic pressure−diastolic systemic pressure. Pulse pressure wascalculated as the difference between systolic and diastolic readings.

After being harvested, the trachea was connected to the cannula of aperfusion system. The left lung was clamped while 5 mL of cold PBSsolution was injected in the trachea to perform bronchoalveolar lavageto obtain fluid (BALF) of the right lobe of the lungs and was collectedfor further analysis (Total cell counts and cytokines measurement). Oncethe BALF sample was collected, it was centrifuged at 1200 rpm for 10minutes at 4° C. The supernatant was frozen at −80° C. until cytokinesanalysis. The cells were then resuspended in PBS and counted with ahemocytometer.

Organ weights were expressed as relative percentages and were calculatedas follows: Relative organ weight=(organ weight×100)/body weight.

Histological Preparation and Categorization: For each rat, the left lobeof the lungs was harvested, perfused and fixed with 10% neutral bufferedformalin. A transversal section of the middle left lobe was cut andforwarded in 10% NBF to the Institute for Research in Immunology andCancer (Montreal, Quebec, Canada). Tissues were embedded, sliced (5-μmthickness), mounted, and conventionally stained. Staining includedhematoxylin and eosin (H&E) and Ashcroft score and finally, a Masson'strichrome staining for the fibrosis quantification. Glass slidescontaining fixed and stained tissues were visualized at 20×magnification (Eclipse T100 microscope, Nikon). At least fivenon-overlapping view fields/lung were selected for microphotographs(Nikon DS-Fi1 digital camera with Nikon NIS Elements 4.30, Nikon).

Alveolar septa and lung structure were estimated using the H&E slides.Tissue was scored with a modified Ashcroft Scale accordingly to themeans of the five non-overlapping view fields previously selected. Glassslide tissue stained with Masson's Trichrome was also visualized using ascanner to determine the percentage of the fibrotic tissue on the slice.

Hydroxyproline Determination: Hydroxyproline (a biomarker for PF)content was determined using a colorimetric assay kit (Cell Biolabs Inc,CA, USA). Part of the right lobe was removed and homogenized in 0.1 mLof water. The supernatant was hydrolyzed in 0.1 mL of 10 N HCl for 6hours at 120° C. Following the addition of 5 mg of activated charcoal,the samples were centrifuged at 10,000 rpm for five minutes, and thesupernatant was transferred to a new tube and processed according to theassay's instructions. The absorbance was measured at 540 nm, and theamount of hydroxyproline was determined and corrected for proteincontent.

Cytokine Quantification: On Day 21, BALF samples were collectedimmediately following exsanguination of the animal. For all groups,one-half of each sample was saved for analysis of the followingcytokines: (1) Macrophage inflammatory protein 1 (MIP1); (2) Monocytechemoattractant protein 1 (MCP1); (3) Interleukin (IL)-6; (4) Interferongamma-induced protein 10 (IP10); and (5) RANTES. The cytokine analysiswas performed using a Luminex assay (Eve Technologies, Calgary,Alberta).

Analysis and Statistics: The primary outcomes involved survival andweight. Additional parameters of note included cardiopulmonary andpressure parameters at surgery, tissue weights, histologic samples,bronchoalveolar pulmonary lavage fluid cell counts, hydroxyprolinelevels, and cytokine. Results are expressed as means SEM. Comparisonswere made on normally distributed data using ANOVA, followed by a Fisherpost hoc test to assess the difference between BLM group with Graph PadPrism Software version 7.0 for Mac (San Diego, Calif., USA). Comparisonsincluded Sham versus BLM and treatment(s) versus BLM. Treatmentdifferences were not compared. Differences were statisticallysignificant when P values were less than 0.05.

Results

Animal Survival and Weights: Of the 19 animals in Groups 2 and 4, thesurvival rate at Day 10 was 82% during this period ((P<0.05, Sham). Incontrast, the survival rate in non-induced and interventional therapy(RPI) animals (Group 1 and Group 3, respectively) was 100% (P<0.05, BLM)(FIG. 2A). At Day 21, the survival rate for the BLM (Group 2) dropped to62% (P<0.05, Sham), whereas preventive treatment (RPT) and RPI survivalrates were 90% and 89.5% (P<0.05, BLM), respectively (FIG. 2B). Animalsin the Sham group continued at 100%.

Mirroring the survival rates were those of body weight (FIG. 2C). Threeweeks after BLM induction, the body weight of those in the Sham groupincreased by approximately 50%; however, the rats in the BLM group wassignificantly lower (P<0.05, Sham). RPT significantly alleviatedBLM-induced weight loss by Day 21 (P<0.01, BLM), as compared with theBLM group. RPT administered from Day 10 following the BLM induction,slightly increased body weight, as compared to BLM-treated rats at Day21.

Hemodynamic and Cardiac Effects: Hemodynamic parameters were recorded onDay 21 (FIG. 3). Animals in the BLM experienced a significant effect(P<0.05, Sham) of the arterial pulse pressure (FIG. 3A) and cardiacoutput (FIG. 2B). Ventricular dysfunction (insufficient left ventricularpreload) and hypovolemia might account for these observations with pulsepressure. Animals in the RPT experienced an improved arterial pulsepressure (P<0.05, BLM), and were at a similar level that was experiencedby those in the Sham group. Additionally, these animals had restoredcardiac output, as compared with those on BLM (P<0.01). RPI slightlyrestored the arterial pulse pressure and cardiac output. Heart rate wasrelatively similar in all groups (FIG. 3C).

Concerning the systemic arterial pressure (FIG. 3 D-F), while nodifferences were noted among the groups, animals in the BLM groupdisplayed a non-significant reduction of systemic arterial pressure;this effect was probably a consequence of the cardiac output reduction.

Parameters Reflective of Pulmonary Fibrosis and Function: Pulmonaryfibrosis is known to induce respiratory resistance; resistance to theair flow through the respiratory tract during inhalation and exhalation.This resistance reduces gas exchange (O₂—CO₂) in the alveolar andcontributes to reducing lifespan. The BLM animals showed a significant(P<0.001, Sham) increase in respiratory resistance (FIG. 4A). Animals inthe RPT group displayed a significant reduction in this parameter(P<0.05, BLM), while those in the RPI group showed improvement (P=0.10,BLM). The hydroxyproline content in the lung was also measured toevaluate the presence of pulmonary fibrosis (FIG. 4B) and reflectedchanges seen in respiratory resistance. The BLM animals had a two-foldhigher hydroxyproline concentration (P<0.05, Sham). Those in the RPT(P<0.05, BLM) and RPI (P<0.01, BLM) groups had a significant diminutionin hydroxyproline concentration.

Reflective of the pulmonary changes induced by BLM, animals in the BLMgroup had significantly (P<0.01, Sham) higher lung weights (FIG. 5A),suggesting the presence of edema. Lung weight of the RPT animals wassignificantly lower (P<0.05, BLM). The lung weight was also decreased inthe RPI group.

Total cell count (inflammation, FIG. 5B) and total protein content(edema, Fib. 5C) were obtained from BALF of the right lobe of the lungsto reinforce lung weight measurements and reflect the presence ofpulmonary edema. Both cell counts and protein levels in the BLM animalsshowed a significant increase (P<0.01, Sham). Animals in the RPT groupshowed a reversal in both cell counts and protein levels (both, P<0.05,BLM). Animals RPI group significantly reduced the total cell counts(P<0.05, BLM) and showed numerical improvement reduction of BALF proteinconcentration.

Staining of lung tissue provided additional evidence reflective of thedevelopment of pulmonary fibrosis with BLM and attenuation with RP5063treatment. As reflected in H&E staining (FIG. 6), the Ashcroft Score inthe BLM group lung tissue samples (FIG. 6 A, C) was significant(P<0.001, Sham). Of the treatments, the samples obtained from RPTanimals displayed a significant reduction in these lung parenchymalfibrotic changes (FIG. 6C), (P<0.001, BLM). Pulmonary fibrosis ischaracterized by excessive collagen disposition in the lung, asreflected by percent collagen areas measured with Masson's trichromestaining. Staining samples from animals in the BLM showed a significant(P<0.001, Sham) increase in the percentage of collagen disposition inthe lung on Day 21 (FIG. 6 B, D). Therapeutic intervention with RP5063(RPT) significantly reduced these changes (P<0.001, BLM). These resultscorrelate nicely with the previously presented hydroxyprolineconcentration quantification.

Reflective of BLM-induced effects on cardiopulmonary capacity, bloodoxygen saturation (FIG. 7A) and blood lactate levels (FIG. 7B) weremeasured at Day 21. The BLM animals showed a significant decrease insaturation and a significant increase in blood lactate levels (both;P<0.01, Sham). Animals in the RPT group showed normalization of theblood oxygen levels (P<0.05, BLM). The saturation levels in RPI animalsimproved and were numerically better than those the BLM group. While thetreatments induced a diminution of blood lactate (a biomarker for PF)levels, both the RPT and RPI were significant (P<0.01, P<0.05,respectively, versus BLM).

BLM-Induced Inflammatory and Fibrogenic Cytokines: To evaluate theimpact of treatment on BLM-induced inflammatory and fibrogeniccytokines, analysis of BALF from Day 21 quantified levels of MIP1, MCP1,IL-6, IP10, and RANTES (FIG. 8A-E). Animals in the BLM group displayed asignificant increase in all the five cytokines levels (P<0.05 for MIP1and MCP1; P<0.01 for IP10 and RANTES, Sham). None of the treatmentsstatistically reversed the production of MIP-1 (FIG. 8A), though RPTslightly reduced the production of this cytokine. RP5063 treated animalgroups showed reductions in MCP-1 concentrations (FIG. 8B) and those inthe RPI group showed a significant decrease (P<0.05, BLM). Animals inboth treatment groups had numerically (RPT, RPI) reduced IL6 levels(FIG. 8C). Animals in both RPT and RPI groups showed significantreductions in IP10 (FIG. 8D) (P<0.01, BLM). Finally, animals in bothRP5063 treated animals had significantly reduced RANTES cytokine levels(FIG. 8E) (P<0.01, BLM).

CONCLUSIONS

In light of the actual criteria for the demonstration of efficacy forIPF treatment, RP5063 (Compound A, brilaroxazine) was able tosignificantly affect all important biomarkers and actual endpointsillustrated in the present study. Treatment with RP5063 attenuatedBLM-induced pulmonary fibrosis, inflammation, and ECM deposition(collagen) and improved cardiac and pulmonary functions in rodents.RP5063, both as a preventive treatment starting Day 1 (RPT) and asinterventional therapy (RPI) beginning on Day 10 followingBLM-induction, was able to reduce IPF progression. Positive effects onbody weight, survival, lung edema, fibrogenic cytokine production,hydroxyproline content, and respiratory resistance, and cardiopulmonarycapacity provide supportive evidence that RP5063 impacts both thefunctional and pathologic effects associated with IPF. The overalleffect in this translational animal model is indicative that RP5063 isan efficacious therapy for the treatment of IPF and PF.

It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims.

What is claimed is:
 1. A method of treating pulmonary fibrosis in asubject, the method comprising administering to a subject suffering frompulmonary fibrosis an effective amount of a compound having thefollowing formula,

or a pharmaceutically acceptable salt, racemate, or diastereomericmixture thereof.
 2. The method according to claim 1, wherein thecompound is in a form of a hydrochloride salt.
 3. The method accordingto claim 1, wherein one or more hydrogens of the compounds areoptionally substituted with ²H (deuterium).
 4. The method according toclaim 1, wherein the compound is administered in a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, excipient,or diluent.
 5. The method according to claim 1, wherein the compound isorally administered.
 6. The method according to claim 1, which treatsidiopathic pulmonary fibrosis.
 7. The method according to claim 1, whichtreats pulmonary fibrosis in a subject with chronic obstructivepulmonary disease (COPD).
 8. The method according to claim 1, whichtreats pulmonary fibrosis in a subject with sickle cell disease (SCD).9. The method according to claim 1, which treats pulmonary fibrosis in asubject with scleroderama.
 10. The method according to claim 1, whichtreats pulmonary fibrosis in a subject with lung cancer.